Direct Photosynthetic Recycling of Carbon Dioxide To Isobutyraldehyde - Assignment Example

Biotechnology Paper Analysis Atsumi et, al. 2009 Questions Detail the photosynthetic pathway in Synechococcus elongatus and indicate where mutations have been introduced. Ans 1. Carbon dioxide fixation in Synechococcus elongates occurs via the Calvin Benson-Bassham Cycle pathway. The enzyme that catalyzes CO2 fixation is Rubisco. It is also the rate limiting step owing to its poor turnover rate and competition of O2 and CO2 at the binding site of the enzyme. The pathway is illustrated as follows: CO2 RuBP 3PGA 2PGA Phospho enol Pyruvate Pyruvate The mutated strains were constructed by inserting the following mutations: Figure a: The ketoacid decarboxylase gene kivd from Lactococcus lactis 13 was expressed using an expression cassette under the control of the isopropyl­ β­D­thiogalactoside (IPTG) inducible promoter Ptrc. This recombination15, resulting in SA578 . Figure b: To increase the flux to the keto acid precursor, 2­ketoisovalerate (KIV)16, the alsS gene from Bacillus subtilis and the ilvC and ilvD genes from Escherichia coli were integrated into neutral site II (NSII)17 of the SA578 genome, resulting in SA590 2. Why do Atsumi et, al. choose to isolate isobutyraldehyde? In this work, isobutyraldehyde was chosen as a target because: 1) it has a low boiling point (63 °C) and a high vapor pressure (66 mm Hg at 4.4 °C), suggesting that it can be readily retrived from microbial cultures during production. 2) Subsequent purification is also relatively easy, and the isobutyraldehyde concentration in the production medium can remain low. 3) Product removal in situ can reduce the cytotoxic effects of isobutyraldehyde and enable long­term production. 4) In addition, isobutyraldehyde can be readily converted to various hydrocarbons currently derived from petroleum (such as isobutanol, isobutyric acid, acetal, oxime and imine) using existing chemical catalysis. In particular, it can be converted either biologically or chemically to isobutanol, which is suitable for use as a gasoline substitute, thus making isobutyraldehyde a useful chemical feedstock. Thus 3. Mathematically show how the productivity (total product divided by volume and time) of isobutyraldehyde compares with biodiesel production? The average productivity of microalgal biodiesel in a well­designed production system can be ~1 × 105 liter ha−1 per year. Assuming that one­third of the day (8 h) and 80% of the calendar year (292 d) are dedicated to production and a characteristic dimension of 1 m for converting areal to volumetric productivity, the productivity of isobutyraldehyde would need to be 3,420 µg l−1 h−1 or higher to be competitive with algal biodiesel production. This level of productivity served as the initial benchmark productivity for direct conversion of CO2 to isobutyraldehyde. Although the productivity of lab­scale experiments cannot be directly translated to industrial­scale production, our productivity of isobutyraldehyde (6,230 µg l−1 h−1) was encouraging 4. (a) Rubisco is believed to be the most abundant protein on earth, where is it found? What does it do? And how does its structure relate to its function. Rubisco turns approximately 10^11 tonnes of carbon every year and provides the only link between pools of inorganic and organic carbon in the biosphere. Yet, with a turnover rate of 3 sec-1 as compared to 1000 molecules sec-1 of other typical enzymes. Rubisco is one of the slowest enzymes known and regulates the rate limiting step in the photosynthetic assimilation of carbon. Plant cells compensate for this slow rate by building lots of the enzyme. Chloroplasts are filled with rubisco, which comprises half of the protein. This makes rubisco the most plentiful single enzyme on the Earth. (b) Comment on the use of the rbcLS gene in this work? Why do they use it and what does it show them? To compensate for the inherent limitations of Rubisco, the rbcLS genes from the related S. elongatus strain PCC6301 were integrated downstream of the endogenous rbcLS genes of SA590, resulting in SA665 . The Rubisco activity of SA665 was found to be ~1.4­fold higher than that of SA590. With the expression of the additional rbcLS genes, SA665 produced 1.1 g/l of isobutyraldehyde over 8 d with a steady state (first 7 d) production rate of 6,230 µg l −1 h−1, which is roughly twofold higher than SA590, which lacks the additional rbcLS genes. The increase in Rubisco activity of SA665 and the concomitant increase in isobutyraldehyde production suggest that CO2 fixation is one of the bottlenecks of the isobutyraldehyde production. Thus, it is possible that the optimization of CO2 fixation could further improve isobutyraldehyde production. To probe the impact of Rubisco over expression, photosynthetic O2 production of SA590 and SA665 were measured under the same conditions as isobutyraldehyde production. The O2 production of SA665 was similar to that of SA590, indicating that overexpression of the rbcLS genes did not enhance the rate of photosynthesis in this condition. Thus, overexpression of Rubisco either allowed more carbon fixation through more efficient utilization of photosynthesis generated reducing power or directed more fixed carbon to isobutyraldehyde. As no other by­product was detected and the cells were not growing, the former explanation appears more plausible. 5. Several genes are expressed using a “Ptrc” promoter, discuss how this promoter is activated and what the requirements for expression from it are? Ptrc is a chemically induced promoter that turns on the gene transcription activity in the presence of the inducer isopropyl­ β­D­thiogalactoside (IPTG). IPTG is a lactose analog which binds and inhibits the lac repressor and thereby strongly induces beta-galactosidase production. Thus Ptrc assists in carbon metabolism in the Calvin cycle. 6. What are the uses of isobutyraldehyde? 1) Isobutyraldehyde can be found naturally in tea, beer, brandy, cigarette smoke, cooked pork and spearmint oil 2) Isobutyraldehyde gas can be transformed into isobutanol and other useful petroleum products by a simple process of catalysis. 3) A compound urea-isobutyraldehyde is used as a feed for animals. 4) Isobutyraldehyde is used as a chemical intermediate in the manufacture of perfumes, flavours and petrol additives. 5) It is used as a solvent (in artificial leather manufacture, textiles and plastics), a component in brake fluid and in the production of pharmaceuticals and vitamins. 7. Discuss the pros and cons of the direct bioconversion of CO2 using photosynthesis in to industrially relevant compounds. Pros: As the author puts it, by direct bioconversion of CO2 using photosynthesis in to industrially relevant compounds may provide an alternative path for addressing two of humanity’s most pressing problems: energy and climate change. It will be almost an ideal method for restoring flow of materials through the natural cycles, avoid bottlenecks and meet the ever increasing energy demands. Since it’s a photosynthetic method the accompanying release of oxygen will also have the much desired cleansing effect on the atmosphere. If the process is viable and standardized it will function like a continuous loop, correcting previous harm and preventing further damage. Cons: Not very apparent though excessive genetic recombination to produce higher bioconverting strains, if leaked might result in weed species. Excess biomass produced also poses disposal problems as it might lead to extremely high BOD and COD demands and thus adequate recycling requirements. References Shota Atsumi, Wendy Higashide, and James C. Liao. Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. Nature biotechnology, 27(12):1177–1180, December 2009. The environment agency < http://www.environment-agency.gov.uk/business/topics/pollution/39199.aspx> Accessed on January 05 2010 by: Shota Atsumi, Wendy Higashide, James C. Liao Atsumi, Shota, Wendy Higashide, and James C. Liao. "Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde.." Nature biotechnology 27 (December 2009): 1177-1180. Read More